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Expression of BMP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells / 南方医科大学学报
Journal of Southern Medical University ; (12): 1383-1388, 2012.
Article in English | WPRIM | ID: wpr-315457
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of bone morphogenetic protein 4 (BMP4) in culturing induced pluripotent stem cells (iPSCs) and the related signal pathways.</p><p><b>METHODS</b>We amplified the mature peptide of BMP4 from the placenta through RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing BMP4 were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell phenotype, cell differentiation capacity into lineages of the 3 germ layers and expression levels of pluripotency-associated genes were investigated.</p><p><b>RESULTS</b>Smad1 was phosphorylated by BMP4 from the culture medium. iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages maintained the phenotype of stem cells with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to differentiate into cell lineages of the 3 germ layers.</p><p><b>CONCLUSION</b>BMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.</p>
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Gene Expression / Cell Differentiation / Cells, Cultured / Immunoglobulin kappa-Chains / Cell Biology / Bone Morphogenetic Protein 4 / Induced Pluripotent Stem Cells / HEK293 Cells / Genetics Limits: Animals / Female / Humans Language: English Journal: Journal of Southern Medical University Year: 2012 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Gene Expression / Cell Differentiation / Cells, Cultured / Immunoglobulin kappa-Chains / Cell Biology / Bone Morphogenetic Protein 4 / Induced Pluripotent Stem Cells / HEK293 Cells / Genetics Limits: Animals / Female / Humans Language: English Journal: Journal of Southern Medical University Year: 2012 Type: Article